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Rationale: SARS-CoV-2 gains entrance to airway epithelial cells (AECs) through binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) on the cell surface. However, ACE2 also converts angiotensin II into angiotensin-(1-7) and counterbalances the renin-angiotensin-aldosterone system, with resultant protective effects in the cardiovascular system. Some data suggest that two common antihypertension medications (angiotensin II receptor antagonists, ARBs; and angiotensin-converting-enzyme inhibitors, ACEIs) may increase ACE2 expression in heart and kidney cells, fueling debate about how these widely used medications may modulate SARS-CoV-2 infectivity and risk of COVID-19.Aim: Determine whether exposure of bronchial AECs to the ARB losartan or the ACEI captopril modulate expression of ACE2 by AECs, SARS CoV2 replication, or expression of proinflammatory cytokines and type I and III interferon (IFN) responses.Methods: Primary bronchial AECs from children and adults (n = 19; Ages 8–75 yrs) were differentiated ex vivo at an air-liquid interface to generate organotypic cultures. Cultures were treated with captopril (1 μM) or losartan (2 μM) with culture media changes starting 72 h before infection with SARS-CoV-2. In a biosafety level 3 (BSL-3) facility, cultures were infected with SARS-CoV-2 isolate USA-WA1/2020 at a multiplicity of infection (MOI) of 0.5. At 96 h following infection, RNA and protein were isolated. SARS-CoV-2 replication in cultures was assessed with quantitative PCR (qPCR). ACE2, IL-6, IL-1B, IFNB1, and IFNL2 expression were assessed by qPCR.Results: Neither captopril nor losartan treatment significantly changed ACE2, IL-6, IL-1B, IFNB1, or IFNL2 expression by AECs as compared to SARS-CoV-2 infected AEC cultures without captopril or losartan treatment. At 96 h following infection, SARS-CoV-2 copy number/ng RNA was not significantly different between untreated AEC cultures, cultures treated with captopril, or cultures treated with losartan.Conclusion: These findings suggest that at the level of the airway epithelium neither the ACEI captopril or ARB losartan significantly modify expression of the SARS-CoV-2 entry factor ACE2, nor does either medication increase replication SARS-CoV-2 replication. This ex vivo data is reassuring and is consistent with evolving clinical data suggesting ACEIs and ARBs do not increase the risk for poor prognosis with COVID-19 and may actually reduce the risk of COVID-19 disease.

Airway remodeling may contribute to decreased lung function in asthmatic children. Bronchial epithelial cells (BECs) may regulate fibroblast expression of extracellular matrix (ECM) constituents and fibroblast-to-myofibroblast transition (FMT). Our objective was to determine if human lung fibroblast (HLF) expression of collagen I (COL1A1), hyaluronan synthase 2 (HAS2), and the FMT marker alpha-smooth muscle actin (α-SMA) by HLFs conditioned by BECs from asthmatic and healthy children correlate with lung function measures and exacerbation history among BEC donors. BECs from asthmatic (n = 23) and healthy children (n = 15) were differentiated at an air-liquid interface (ALI) and then co-cultured with HLFs for 96 hours. Expression of COL1A1, HAS2, and α-SMA by HLFs was determined by quantitative polymerase chain reaction (qPCR). FMT was quantified by measuring HLF cytoskeletal α-SMA by flow cytometry. Pro-collagen Iα1, hyaluronan (HA), and PGE2 were measured in BEC-HLF supernatant. Correlations between lung function measures of BEC donors, and COL1A1, HAS2, and α-SMA gene expression, as well as supernatant concentrations of HA, pro-collagen Iα1, hyaluronan (HA), and PGE2 were assessed. We observed that expression of α-SMA and COL1A1 by HLFs co-cultured with asthmatic BECs was negatively correlated with BEC donor lung function. BEC-HLF supernatant concentrations of pro-collagen Iα1 were negatively correlated, and PGE2 concentrations positively correlated, with asthmatic BEC donor lung function. Expression of HAS2, but not α-SMA or COL1A1, was greater by HLFs co-cultured with asthmatic BECs from donors with a history of severe exacerbations than by HLFs co-cultured with BECs from donors who lacked a history of severe exacerbations. In conclusion, α-SMA and COL1A1 expression by HLFs co-cultured with BECs from asthmatic children were negatively correlated with lung function measures, supporting our hypothesis that epithelial regulation of HLFs and airway deposition of ECM constituents by HLFs contributes to lung function deficits among asthmatic children. Furthermore, epithelial regulation of airway HAS2 may influence the susceptibility of children with asthma to experience severe exacerbations. Finally, epithelial-derived PGE2 is a potential regulator of airway FMT and HLF production of collagen I that should be investigated further in future studies.

Background Airway inflammation is a hallmark of asthma. Alterations in extracellular matrix (ECM) hyaluronan (HA) content have been shown to modulate the recruitment and retention of inflammatory cells. Bronchial epithelial cells (BECs) regulate the activity of human lung fibroblasts (HLFs); however, their contribution in regulating HLF production of HA in asthma is unknown. In this study, we tested the hypothesis that BECs from asthmatic children promote the generation of a pro-inflammatory, HA-enriched ECM by HLFs, which promotes the retention of leukocytes. Methods BECs were obtained from well-characterized asthmatic and healthy children ages 6–18 years. HLFs were co-cultured with BECs for 96 h and samples were harvested for analysis of gene expression, synthesis and accumulation of HA, and subjected to a leukocyte adhesion assay with U937 monocytes. Results We observed increased expression of HA synthases HAS2 and HAS3 in HLFs co-cultured with asthmatic BECs. Furthermore, we demonstrated greater total accumulation and increased synthesis of HA by HLFs co-cultured with asthmatic BECs compared to healthy BEC/HLF co-cultures. ECM generated by HLFs co-cultured with asthmatic BECs displayed increased HA-dependent adhesion of leukocytes in a separate in vitro binding assay. Conclusions Our findings demonstrate that BEC regulation of HA production by HLFs is altered in asthma, which may in turn promote the establishment of a more leukocyte-permissive ECM promoting airway inflammation in this disease. Electronic supplementary material The online version of this article (10.1186/s12931-018-0849-1) contains supplementary material, which is available to authorized users.

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.

Background An increasing number of studies using primary human bronchial epithelial cells (BECs) have reported intrinsic differences in the expression of several genes between cells from asthmatic and non-asthmatic donors. The stability of gene expression by primary BECs with increasing cell passage number has not been well characterized. Methods To determine if expression by primary BECs from asthmatic and non-asthmatic children of selected genes associated with airway remodeling, innate immune response, immunomodulatory factors, and markers of differentiated airway epithelium, are stable over increasing cell passage number, we studied gene expression patterns in passages 1, 2, 3, 4, and 5 BECs from asthmatic (n = 6) and healthy (n = 6) subjects that were differentiated at an air-liquid interface. RNA was harvested from BECs and RT-PCR was performed for TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, TSLP, IL-33, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1. Results Expression of TGFβ1, TGFβ2, activin A, FSTL3, MUC5AC, CXCL10, IFIH1, p63, KT5, TUBB4A, TJP1, OCLN, and FOXJ1 by primary BECs from asthmatic and healthy children was stable with no significant differences between passages 1, 2 and 3; however, gene expression at cell passages 4 and 5 was significantly greater and more variable compared to passage 1 BECs for many of these genes. IL-33 and FOXJ1 expression was also stable between passages 1 through 3, however, expression at passages 4 and 5 was significantly lower than by passage 1 BECs. TSLP, p63, and KRT5 expression was stable across BEC passages 1 through 5 for both asthmatic and healthy BECs. Conclusions These observations illustrate the importance of using BECs from passage ≤3 when studying gene expression by asthmatic and non-asthmatic primary BECs and characterizing the expression pattern across increasing cell passage number for each new gene studied, as beyond passage 3 genes expressed by primary BECs appear to less accurately model in vivo airway epithelial gene expression. Electronic supplementary material The online version of this article (10.1186/s12890-018-0652-2) contains supplementary material, which is available to authorized users.

The molecular networks underlying the lung response to hypoxia are not fully understood. We employed systems biology approaches to study temporal effects of intermittent or sustained hypoxia on gene expression in rat lungs. We obtained gene expression profiles from rats exposed to intermittent or sustained hypoxia lasting 0–30 days and identified differentially expressed genes, their patterns, biological processes, and regulatory networks critical for lung response to intermittent or sustained hypoxia. We validated selected genes with quantitative real-time PCR. Intermittent and sustained hypoxia induced two distinct sets of genes in rat lungs that displayed different temporal expression patterns. Intermittent hypoxia induced genes mostly involved in ion transport and homeostasis, neurological processes, and steroid hormone receptor activity, while sustained hypoxia induced genes principally participating in immune responses. The intermittent hypoxia-activated network suggested a role for cross talk between estrogen receptor 1 (ESR1) and other key proteins in hypoxic responses. The sustained hypoxia-activated network was indicative of vascular remodeling and pulmonary hypertension. We confirmed the temporal expression changes of 12 genes (including the Esr1 gene and 4 ESR1 target genes) in intermittent hypoxia and 8 genes in sustained hypoxia with quantitative real-time PCR. Conclusions: intermittent and sustained hypoxia induced distinct gene expression patterns in rat lungs. The functional characteristics of genes activated by these two distinct perturbations suggest their roles in the downstream physiological effects of intermittent and sustained hypoxia. Our results demonstrate the discovery potential of applying systems biology approaches to the understanding of mechanisms underlying hypoxic lung response.

Background Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs. Methods BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin (α-SMA) and flow cytometry was used to assay for α-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments, we investigated the role of TGFβ2 in BEC-HLF co-cultures using monoclonal antibody inhibition. Results Expression of α-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs, but not different than α-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less α-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGFβ2 led to similar expression of α-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone. Conclusion These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGFβ2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling.